OBJECTIVE:To investigate the effects of Huayu Qutan recipe(化瘀祛痰方,HYQT)on the atherosclerosis(AS)model of ApoE-/-mice with a high-fat diet and to illustrate the underlying mechanisms from modern pathophysiological...OBJECTIVE:To investigate the effects of Huayu Qutan recipe(化瘀祛痰方,HYQT)on the atherosclerosis(AS)model of ApoE-/-mice with a high-fat diet and to illustrate the underlying mechanisms from modern pathophysiological conceptualizations.METHODS:High performance liquid chromatography of quadrupole time of flight-tandem mass spectrometry(HPLC-Q-TOF-MS/MS)analysis was used to identify the active compounds in the recipe.The mice were randomly allocated into 7 groups:control(CTRL)group,normal diet(ND)group,high-fat diet(HFD)group,HYQT groups(low dose,medium dose,and high dose),and simvastatin(SIM)group.Deferent doses of HYQT were gavaged twice a day,and then the protective effect of HYQT on plaque formation in ApoE-/-mice with a high-fat diet was verified via hematoxylin-eosin(HE)staining and oil red o(ORO)staining.We observed the co-localization in aortic macrophages and lipid droplets(LDs)by CD68 and the Bodipy fluorescence probe.Light chain 3 phosphoprotein classⅡ/light chain 3 phosphoprotein classⅠ(LC3Ⅱ/LC3Ⅰ)was examined by western blotting,and sequestosome 1(SQSTM1/p62),Beclin1,Lamp1,mammalian target of rapamycin(mTOR),phosphorylated mammalian target of rapamycin(p-mTOR),and ATPbinding cassette transporter A1(ABCA1)were examined by real-time polymerase chain reaction(RT-PCR)and Western blotting.Transcription factor EB(TFEB)nuclear translocation was determined by immunofluorescence analysis.RESULTS:Five active compounds were identified using HPLC-Q-TOF-MS/MS analysis:ferulic acid,chlorogenic acid,calycosin,formononetin,and 8,2'-dihydroxy-7,4'-dimethoxy-isoflavane.The effect of HYQT on atherosclerotic plaque formation in Apo E-/-mice was investigated.These findings showed that HYQT decreased the co-localization of CD68 and Bodipy and increased the co-localization of CD68 and LC3B.Medium and high doses of HYQT increased autophagosome formation and promoted the maturation of LC3Ⅱ/LC3Ⅰ.Additionally,HYQT decreased the expression of SQSTM1/p62.Medium and high doses of HYQT also increased the expression of Beclin1 and Lamp1.RT-PCR and Western blot results suggested that HYQT enhanced the expression of ABCA1 mRNA and protein and regulated the mTORC1/TFEB signaling pathway.CONCLUSION:The results indicate that HYQT is an effective traditional Chinese herbal remedy for the treatment of AS.HYQT mitigates macrophage-derived foam cell formation by activating autophagy in atherosclerosis.The m TOR/TFEB signaling pathway and ABCA1 are therapeutic targets of HYQT for the treatment of AS.展开更多
基金the Science and Technology Research Project of Education Department of Liaoning Province:the Mechanism of Regulating Lipophagy and Affecting Cholesterol Efflux in Human Promyelocytic Leukemia Cell Line Macrophages by Huayu Qutan Recipe based on the Theory of"Laoxia Shengtan"(No.L202048)Youth Project of Basic Scientific Research Project of Education Department of Liaoning Province:Mechanism of Mitochondrial Autophagy and Apoptosis in L02 Hepatocytes Regulated by miR-7043-3p based on the Theory of"Spleen Qi Dispersing Essence"of Huayu Qutan Recipe(No.LJKQZ2021064)。
文摘OBJECTIVE:To investigate the effects of Huayu Qutan recipe(化瘀祛痰方,HYQT)on the atherosclerosis(AS)model of ApoE-/-mice with a high-fat diet and to illustrate the underlying mechanisms from modern pathophysiological conceptualizations.METHODS:High performance liquid chromatography of quadrupole time of flight-tandem mass spectrometry(HPLC-Q-TOF-MS/MS)analysis was used to identify the active compounds in the recipe.The mice were randomly allocated into 7 groups:control(CTRL)group,normal diet(ND)group,high-fat diet(HFD)group,HYQT groups(low dose,medium dose,and high dose),and simvastatin(SIM)group.Deferent doses of HYQT were gavaged twice a day,and then the protective effect of HYQT on plaque formation in ApoE-/-mice with a high-fat diet was verified via hematoxylin-eosin(HE)staining and oil red o(ORO)staining.We observed the co-localization in aortic macrophages and lipid droplets(LDs)by CD68 and the Bodipy fluorescence probe.Light chain 3 phosphoprotein classⅡ/light chain 3 phosphoprotein classⅠ(LC3Ⅱ/LC3Ⅰ)was examined by western blotting,and sequestosome 1(SQSTM1/p62),Beclin1,Lamp1,mammalian target of rapamycin(mTOR),phosphorylated mammalian target of rapamycin(p-mTOR),and ATPbinding cassette transporter A1(ABCA1)were examined by real-time polymerase chain reaction(RT-PCR)and Western blotting.Transcription factor EB(TFEB)nuclear translocation was determined by immunofluorescence analysis.RESULTS:Five active compounds were identified using HPLC-Q-TOF-MS/MS analysis:ferulic acid,chlorogenic acid,calycosin,formononetin,and 8,2'-dihydroxy-7,4'-dimethoxy-isoflavane.The effect of HYQT on atherosclerotic plaque formation in Apo E-/-mice was investigated.These findings showed that HYQT decreased the co-localization of CD68 and Bodipy and increased the co-localization of CD68 and LC3B.Medium and high doses of HYQT increased autophagosome formation and promoted the maturation of LC3Ⅱ/LC3Ⅰ.Additionally,HYQT decreased the expression of SQSTM1/p62.Medium and high doses of HYQT also increased the expression of Beclin1 and Lamp1.RT-PCR and Western blot results suggested that HYQT enhanced the expression of ABCA1 mRNA and protein and regulated the mTORC1/TFEB signaling pathway.CONCLUSION:The results indicate that HYQT is an effective traditional Chinese herbal remedy for the treatment of AS.HYQT mitigates macrophage-derived foam cell formation by activating autophagy in atherosclerosis.The m TOR/TFEB signaling pathway and ABCA1 are therapeutic targets of HYQT for the treatment of AS.
